The cellular processes employed by phagocytic cells to neutralize and remove invasive microorganisms include diverse biochemical mechanisms which attack vital elements of the microbial target. Among the neutrophil's microbicidal tools are antibiotic peptides, stored in cytoplasmic granules for delivery into the phagolysosome where the peptides contribute to the efficient killing process. Within the last decade, a remarkable number of potent, broad-spectrum microbicidal peptides have been isolated, revealing both the structural diversity and the evolutionary importance of this class of host defense molecules. In this application, experiments are proposed which will further our understanding of the structures and molecular mechanisms of three classes of neutrophil antimicrobial peptides: defensins, beta-defensins, and indolicidin. With tools developed during the last grant period, a multidisciplinary approach will be employed to delineate the molecular mechanism(s) of these unique host defense molecules. Three interactive specific aims are proposed: 1) using synthetic and recombinant methods, mutant peptides will be produced in order to test specific hypotheses regarding the conformational requirements and molecular interactions which underlie the killing process; these studies will seek to determine the role of dimerization in peptide-mediated killing, as well as the contribution of amino-terminal conformations; 2) to refine our understanding of the relationships between molecular structure and antimicrobial activity, additional studies will be undertaken to determine the solution and crystal structures of selected natural, mutant, and chimeric peptides: indolicidin, and peptides selected from the defensin and beta-defensin families will be synthesized or produced by heterologous expression to provide material for these studies; 3) studies on peptide- bilayer interactions will be expanded by conducting experiments to more precisely characterize mechanisms of membrane permeabilization, as well as the nature of the molecular assemblies involved in pore-formation; experiments will be conducted to measure peptide binding and lysis of unilamellar vesicles, and to characterize the orientation of the peptides in phospholipid multilayers using small angle X-ray diffraction. The results of these investigations will provide new insight into the role and mechanisms of endogenous antibiotic peptides in host defense. These in turn may have value in applied fields, as in the rational design of novel therapeutic agents.